Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the release of immunoreactive somatostatin (IR-SRIF) from hypothalamic cells that were obtained from rats and dispersed with the aid of collagenase. Twenty-four hours after dispersion, cells were placed in a column supported by a matrix of preswollen Biogel P2 and perifused. Fractions were collected on ice and subsequently assayed for SRIF. SRIF release was stimulated markedly by potassium depolarization (KCl, 56 mM), by the Na+-K+-ATPase inhibitor ouabain (10(-4) M), and by dopamine at concentrations as low as 10(-11) M. The stimulatory effects of membrane depolarization were calcium dependent and were not observed in the absence of exogenous calcium in the perifusion medium or in the presence of EDTA (0.05 M). Metoclopramide, the dopamine antagonist, abolished the stimulatory effect of dopamine. In conclusion, release of IR-SRIF by dispersed rat hypothalamic cells can be studied in a simple perifusion apparatus. Release is stimulated by membrane depolarization in a calcium-dependent manner and by dopamine at physiological concentrations.
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PMID:Dopamine stimulates somatostatin release from perifused hypothalamic cells. 613 72

Nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.8) activity is associated with matrix vesicles purified from collagenase digests of fetal calf epiphyseal cartilage. This enzyme hydrolyzes nucleoside triphosphates to nucleotides and PPi, the latter inducing precipitation in the presence of Ca2+ and Pi. An assay for matrix vesicle nucleoside triphosphate pyrophosphohydrolase is developed using beta, gamma-methylene ATP as substrate. The assay is effective in the presence of matrix vesicle-associated ATPase, pyrophosphatase, and alkaline phosphatase activities. A soluble nucleoside triphosphate pyrophosphohydrolase is obtained from matrix vesicles by treatment with 5 mM sodium deoxycholate. The solubilized enzyme induced the precipitation of calcium phosphate in the presence of ATP, Ca2+, and Pi. Extraction of deoxycholate-solubilized enzymes from matrix vesicles with 1-butanol destroys nucleoside triphosphate pyrophosphohydrolase activity while enhancing the specific activities of ATPase, pyrophosphatase, and alkaline phosphatase. In solutions devoid of ATP and matrix vesicles, concentrations of PPi between 10 and 100 microM induce calcification in mixtures containing initial Ca2+ X P ion products of 3.5 to 7.9 mM2. This finding plus the discovery of nucleoside triphosphate pyrophosphohydrolase in matrix vesicles supports the view that these extracellular organelles induce calcium precipitation by the enzymatic production of PPi. Nucleoside triphosphate pyrophosphohydrolase is more active against pyrimidine nucleoside triphosphates than the corresponding purine derivatives. The pH optimum is 10.0 and the enzyme is neither activated nor inhibited by Mg2+ or Ca2+ ions or mixtures of the two. Vmax at pH 7.5 for beta, gamma-methylene ATP is 0.012 mumol of substrate hydrolyzed per min per mg of protein and Km is below 10 microM. The enzyme is irreversibly destroyed at pH 4 and is stable at pH 10.5.
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PMID:The role of nucleoside triphosphate pyrophosphohydrolase in in vitro nucleoside triphosphate-dependent matrix vesicle calcification. 613 31

Rat pancreases were minced and treated with collagenase or collagenase supplemented with chymotrypsin to yield a mixture of ducts, islets, acinar cell clusters, blood vessels, and nerves. Histologically and ultrastructurally, the isolated tissues resembled their in situ counterparts in most respects, the major difference being the destruction of the basement membranes (basal laminae). Ducts ranging in size from the common bile/main pancreatic duct to the intercalated ducts were identified in the digest, although interlobular ducts were most frequently observed. Acinar tissue fragments were separated from nonacinar structures either by flotation through discontinuous gradients of Ficoll or by sieving, the latter technique being the more efficient. Common bile/main ducts, interlobular ducts, and blood vessels were selected manually from the nonacinar fractions. Biochemical analyses showed that the entire nonacinar fraction, as well as isolated ducts and blood vessels, contained larger alkaline phosphatase, carbonic anhydrase, and Mg-ATPase specific activities than acinar tissue, whereas acinar tissue contained larger gamma-glutamyltranspeptidase and amylase activities. However, greater than 63% of the total recovered activity of each enzyme was associated with the acinar tissue. Both the association of the majority of each of these enzyme activities with the acinar tissue and the similarity in specific activities associated with ducts and blood vessels indicate that none of the enzymes tested is a unique marker for interlobular and larger ducts of the pancreas of the rat.
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PMID:Characterization of ducts isolated from the pancreas of the rat. 615 56

Rat and hamster pancreatic ducts were isolated by digestion with collagenase plus chymotrypsin and were cultured for eight weeks in an agarose matrix. Freshly isolated and cultured ducts were characterized morphologically and biochemically. The in vivo morphology of the ducts was maintained in vitro, although certain differences were noted. Both interlobular and intralobular ducts could be identified. gamma-Glutamyltranspeptidase and Mg-ATPase were stable enzymatic activities of the ducts of both species; alkaline phosphatase persisted only in the hamster ducts. Carbonic anhydrase and (Na + K)ATPase were minor activities of the rat ducts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the rat ducts suggested that actin was the major duct peptide and that the major zymogens were greatly diminished. These results demonstrate that pancreatic ducts can be maintained in vitro and can be used for biochemical studies of this minor pancreatic tissue type.
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PMID:Morphologic and biochemical characteristics of isolated and cultured pancreatic ducts. 616 52

Extracellular matrix vesicles from bone and epiphyseal cartilage of femur and tibia of rats were isolated by collagenase digestion (crude vesicles) and further purified by sucrose gradient centrifugation. Fractions containing cells and membranes were also isolated from the two tissues. The alkaline and acid phosphatase and ATPase activities, as well as protein content of all fractions including crude and purified matrix vesicles, were assayed. The crude vesicles from both tissues demonstrated a high alkaline phosphatase specific activity (5-20 times higher than in the cell fraction). The total enzyme activities and protein content were significantly higher in all fractions from cartilage than those from bone. A major peak of alkaline phosphatase activity and protein content was obtained following the sucrose gradient centrifugation. The position of this peak was similar for both tissues. The specific activity of alkaline phosphatase of purified matrix vesicles was significantly higher in bone than in cartilage. The phosphatase activities from cartilage and bone showed a similar pH dependence and a similar response to metal ions. Of the metal ions tested (Na+, Mg2+, Zn2+, and Ca2+) only Zn2+ (at 5 mM concentration) inhibited significantly the alkaline phosphatase activity of purified matrix vesicles. The electrophoretic profile of purified matrix vesicles showed eight major protein bands common for both tissues. In addition, cartilage vesicles appeared to possess two peptides not found in bone.
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PMID:Biochemical characterization of matrix vesicles from bone and cartilage. 623 53

Myoepithelial and secretory cells from the mammary gland of the lactating rat have been isolated, purified, and characterized. Mammary tissue was dissociated with collagenase into basket-like networks of myoepithelial cells and single secretory cells. Because of their larger size, the myoepithelial cell networks could be separated from other mammary and blood cells by differential centrifugation. Isolated secretory cells were purified by isopycnic centrifugation in 25% bovine serum albumin. The purified myoepithelial and secretory cells were viable, as shown by the incorporation of 32P into distinct macromolecules that were separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both myoepithelial and secretory cells retained their characteristic morphology after isolation and purification, as shown by light, transmission, and scanning electron microscopies. The isolated myoepithelial cells were unique and, thus, distinguishable from other mammary cells in a number of respects; they 1) contracted in response to the addition of oxytocin, 2) bound [3H]oxytocin specifically, 3) accounted for the content of alkaline phosphatase and [Na+ + K+]ATPase in mammary tissue, and 4) reacted specifically with antiserum prepared against purified myoepithelial cells. The purified secretory cells were unique in possessing glucose-6-phosphate dehydrogenase activity. The different cell markers not only gave independent estimates of the purity of the cell fractions, but they also may be helpful in identifying mammary cells in stages of differentiation and neoplastic transformation.
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PMID:Purification and characterization of mammary myoepithelial and secretory cells from the lactating rat. 624 56

Isolated myocytes were prepared from adult canine hearts using a combined technique of myocardial perfusion followed by incubation with collagenase. More than 60% of the cells routinely excluded trypan blue dye. Disruption of the myocytes was accomplished using high pressure nitrogen cavitation. After differential and sucrose gradient centrifugation, the peak sarcolemmal fraction averaged 100-fold enrichment in ouabain-inhibited K+-stimulated p-nitrophenyl phosphatase and 82-fold in ouabain-inhibited (Na+,K+)-ATPase. These sarcolemmal membranes are enriched in phospholipid phosphorus (1.98 mumol/mg of protein) and more than 4-fold in sphingomyelin and cholesterol. Polyacrylamide gels revealed three major protein peaks at 50,000, 91,000, and 140,000 apparent molecular weights. This work demonstrates the feasibility of preparing highly pure cardiac sarcolemma from isolated adult myocytes. The problem of cellular cross-contamination due to heterogeneity of cell types in whole myocardial tissue has been circumvented. The level of enrichment exceeds all reported preparations of cardiac sarcolemma from whole myocardium and cultured myocytes. This preparation should prove to be useful as an in vitro model for studies of physiological, pharmacological, and pathological perturbations of sarcolemmal structure and function.
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PMID:Preparation and properties of highly enriched cardiac sarcolemma from isolated adult myocytes. 624 86

A homogeneous population of single cells from the thick ascending limb of Henle's loop (TALH) has been isolated from the rabbit kidney medulla. A total medullary cell suspension was prepared by a series of collagenase, hyaluronidase, and trypsin digestions and separated on a Ficoll gradient (2.6-30.7% wt/wt). Morphologically, the cells isolated from the TALH were homogeneous and showed polarity within their plasma membrane structure, with a few blunt microvilli on their apical surface and deep infoldings of the basal-lateral membrane. Biochemically, the TALH cells were highly enriched in calcitonin-sensitive adenylate cyclase and Na, K-ATPase. Alkaline phosphatase and arginine vasopressin-sensitive adenylate cyclase, highly concentrated in proximal tubule and collecting duct, were present only in low concentrations in the TALH cells. Additionally, furosemide, a diuretic inhibiting sodium chloride transport in the TALH in vivo, inhibited oxygen consumption of the TALH cells in a dose-dependent manner. The TALH cells were viable, as judged by morphological appearance, trypan blue exclusion, the response of oxygen consumption to 2,4-dinitrophenol, succinate and ouabain, and the cellular Na, K and ATP levels.
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PMID:Separation of renal medullary cells: isolation of cells from the thick ascending limb of Henle's loop. 625 27

The salt glands of control and salt-stressed ducklings were dissociated with collagenase to produce cell aggregates or 'minilobules' which were cultured. The fine-structural organization of freshly isolated and cultured (up to 72 h) aggregates were examined and showed surprisingly intact fine-structural organization with minimal changes from untreated glands. Incorporation of [3H]leucine into total protein, membrane protein and immunoprecipitable Na,K ATPase was determined in cultures at various time points, by pulse or pulse-chase experiments. Incorporation of label was linear for 4 h in protein, but higher in cultures of salt-stressed glands than in controls. Na,K ATPase was synthesized throughout the time of the experiment, the rate being highest during the first 4 h, reaching a plateau by 24 h. Up to 10% of the total label was present in Na,K ATPase. These results are discussed with reference to the use of minilobule culture to analyse further synthesis and assembly during biogenesis and control of these processes.
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PMID:Organotypic cultures of the avian salt gland: biosynthesis of membrane proteins. 626 44

The enzymatic activity of bone matrix vesicles from parathyroidectomized rats was determined and compared to the activity of vesicles from sham operated and normal animals. The vesicles were isolated from the alveolar bone by collagenase digestion and differential centrifugation and further purified on a discontinuous sucrose density gradient. The amount of extractable protein and the activity of alkaline phosphatase, acid phosphatase, and ATPase in the vesicle fractions thus obtained did not differ significantly from the values characteristic of preparations from control rats. It may therefore be suggested that parathyroid hormone depletion and the associated hypocalcemia have no significant effect on the occurrence and phosphatase activity of bone matrix vesicles.
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PMID:Extracellular matrix vesicles in rat bone after parathyroidectomy. 629 69


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